UK-1 analogues: methods of preparation and use

ABSTRACT

The present invention includes a number of structural analogues of UK-1. A comparision of the anticancer activity of the UK-1 analogues with their ability to inhibit the growth of methicillin-sensitive and methicillin-resistant  Staphylococcus aureus  demonstrates that a structurally simplified analogue of UK-1 retains the natural product&#39;s selective activity against cancer cells. Structurally conservative changes to UK-1 that diminish Mg 2+ -binding ability may result in a dramatic decrease in cancer cell cytotoxicity. The results may establish a minimum structural pharmacophore as well as a functional role for Mg 2+ -binding in the selective cytotoxicity of UK-1.

PRIORITY CLAIM

This application claims priority to Provisional Patent Application No. 60/428,379 entitled “UK-1 ANALOGUES: METHODS OF PREPARATION AND USE” filed on Nov. 22, 2002.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention generally relates to bis(benzoxazole) compositions and methods of making the compositions. More particularly, the invention relates to analogues of natural occurring bis(benzoxazole) compositions and methods of making bis(benzoxazole) analogues.

2. Description of the Relevant Art The relatively simple yet novel bis(benzoxazole) natural products UK-1 and AJ19561 represent a new class of cytotoxic secondary metabolites. Interest in this class of bis(benzoxazoles) is fueled by the finding that UK-1 is selectively cytotoxic to cancer cells but not bacterial cells, yeast, or fungi. This selective cytotoxicity may be mediated through the specific interaction of UK-1 with an as yet undefined cancer cell-associated target. Bis(benzoxazole) natural products are a structurally unique class of Streptomyces secondary metabolites that have recently been reported in the literature. Research groups have isolated UK-1 from acetone extracts of Streptomyces sp. 517-02.

Research groups have isolated AJI9561 from Streptomyces sp. AJ956.

UK-1 and AJI9561 were reported to possess growth inhibitory properties against murine cancer cell line P388, with IC₅₀ values from 0.3-1.6 μM. While UK-1 has cancer cell cytotoxic properties, UK-1 does not inhibit the growth of gram-positive or gram negative bacteria, yeast, or fungi at concentrations as large as 250 μM.

The selective cytotoxicity of UK-1 towards cancer cells versus bacteria and fungi indicates that UK-1 may have a unique mechanism of anticancer action. The 2-(2′-hydroxyphenyl) benzoxazole moiety present in UK-1 is also present in a number of synthetic metal ion chelators and is analogous to the 2-(2′-hydroxyphenyl)oxazole moiety present in a class of microbial siderophores.

The cytotoxicity of UK-1 against P388, B 176, and HeLa cell lines have been previously reported.

Cytotoxicity of UK-1 against Cancer Cell Lines.

Cell Line IC₅₀ ^(a) MCF-7  1.6 μM HT-29   65 μM HL60 0.32 μM PC-3  0.4 μM MDA-231  0.5 μM BT-20 0.17 μM DU145  0.2 μM SKBR3  0.1 μM A549  1.9 μM SK-N-AS   24 μM SK-N-D7 0.17 μM SK-N-F1   7 μM SK-N-MC  1.1 μM SK-N-SH   7 μM CHP-212   12 μM IMR-32 0.06 μM NGP 0.02 μM ^(a)determined using the alamarBlue assay. See Examples for details. UK-1 displays a wide spectrum of potent anticancer activity against leukemia, lymphoma, and certain solid tumor-derived cell lines. In particular, certain neuroblastoma cell lines (IMR-32 and NGP) were very sensitive to UK-1, with IC₅₀ values less than about 100 nM. Other cell lines, such as colon (HT-29), were less sensitive. Despite the potential anticancer activity, there was no indication of any antibacterial effect against Staphylcoccus aureus, methicillin-resistant S. aureus, or Pseudomonas aeruginosa at concentrations up to 50 μg/mL of UK-1.

The metal ion binding ability of synthetically prepared UK-1 has also been investigated. The studies indicate that UK-1 is capable of binding a variety of biologically important metal ions, of particular interest, Mg²⁺ ions. UK-1 binds DNA in a metal ion-dependent fashion like the Mg²⁺ binding aureolic acid group of antitumor antibiotics and synthetic antitumor quinobenzoxazines. One consequence of the interaction of UK-1 with DNA is the inhibition of topoisomerase II.⁹ A more recent investigation of the metal mediated double strand DNA (ds) binding by Electrospray Ionization Mass Spectrometry (ESI-MS) demonstrated that UK-1 forms complexes of the type [ds+UK-1+M²⁺] with a variety of metals, such as Ni²⁺, Co²⁺, and Zn²⁺. The results distinguish the metal mediated DNA binding of UK-1 with the observed metal mediated DNA binding of aureolic acids and quinobenzoxazines. Aureolic acids form metal mediated DNA binding complexes of the form [ds+2× ligand+M²+]. Quinobenzoxazines bind as [ds+2× ligand+2×M2+] metal mediated DNA binding complexes.

The semi-synthetic derivatives methyl UK-1 (MUK-1) and demethyl UK-1 (DMUK-1) both have shown activity against Gram-positive and Gram-negative bacteria. MUK-1 also is active against yeast and filamentous fungi.

SUMMARY OF THE INVENTION

A series of analogues of the bis(benzoxazole) natural product UK-1 in which the carbomethoxy-substituted benzoxazole ring of the natural product was modified were prepared. Some UK-1 analogues may have important anticancer and antibacterial properties. An analogue of UK-1 in which the carbomethoxy-substituted benzoxazole ring was replaced with a carbomethoxy-substituted benzimidazole ring was inactive against human cancer cell lines and the two strains of S. aureus. In contrast, a simplified analogue in which the carbomethoxy-substituted benzoxazole ring was replaced with a carbomethoxy group was almost as active as UK-1 against the four cancer cell lines examined, but lacked activity against S. aureus. Metal ion binding studies of the UK-1 analogues demonstrate that they both bind Zn²⁺ and Ca ions about as well as UK-1. The non-cytotoxic benzimidazole UK-1 analogue binds Mg²⁺ ions 50-fold weaker than UK-1, whereas the simple benzoxazole analogue binds Mg²⁺ ions nearly as well as UK-1. Mg²⁺ ion binding may play a role in the selective cytotoxicity of UK-1 and may provide a minimal pharmacophore for the selective cytotoxic activity of the natural product.

BRIEF DESCRIPTION OF THE DRAWINGS

Other objects and advantages of the invention will become apparent upon reading the following detailed description and upon reference to the accompanying drawings in which:

FIG. 1 depicts an embodiment of a method of forming compound 2.1;

FIG. 2 depicts an embodiment of a method of forming compound 2.9;

FIG. 3 depicts an embodiment of a method of forming compound 2.13;

FIG. 4 depicts an embodiment of a method of forming compound 2.15; and

FIG. 5 depicts cleavage of supercoiled DNA by UK-1 in the presence of oxidizing agents and metal ions.

While the invention is susceptible to various modifications and alternative forms, specific embodiments thereof are shown by way of example in the drawings and will herein be described in detail. It should be understood, however, that the drawing and detailed description thereto are not intended to limit the invention to the particular form disclosed, but on the contrary, the intention is to cover all modifications, equivalents and alternatives falling within the spirit and scope of the present invention as defined by the appended claims.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

A number of UK-1 analogues were prepared in which the carbomethoxy-substituted benzoxazole ring was either modified or deleted. In some embodiments, the synthesis of UK-1 analogues was carried out in analogy with the published synthesis technique for the natural product UK-1, with some improvements. The techniques were published in the paper “The total synthesis of UK-1” to DeLuca and Kerwin Tetrahedron Lett. 1997, 38, 199-202, which is incorporated herein by reference.

A UK-1 analogue may be of the general structure, I:

In some embodiments, A is a substituent of the form, Ia:

In some embodiments, A is a substituent of the form, Ib:

B may be CO₂R³, CON(R⁴)₂, 2-benzoxazoyl, 2-benzothiazoyl group optionally substituted with CO₂R, CON(R ⁴)₂, and/or any other group that imparts Mg²⁺ binding ability. Each occurance of R, R¹, R², R³, R⁴ may be independently, but is not limited to: H, alkyl, halo, haloaklyl, alkoxy, alkylamino, amino acid, peptide, COOR⁴, and/or COR⁴. In some embodiments, n may be between 0 and 4. In some embodiments, X may be NR³. X may be O and/or S. In an embodiment, Y is CH. Y may be CR² and/or N.

In some embodiments, A is a substituent of the form, Ia:

wherein Y is CH, CR¹, or N;

wherein n is 1 to 3; and

wherein each R¹ is independently H, alkyl, halo, haloalkyl, alkoxy, arylalkyl, or alkylamino, and wherein R¹ is positioned on any carbon ortho, meta, or para to the —OH group. In one embodiment, R¹ is a benzyl group in an ortho position to with respect to the —OH group.

In an embodiment, the UK-1 analogue, compound 2.1, is formed when a substituted benzimidazole ring replaces the carbomethoxy-substituted benzoxazole ring of UK-1. In some embodiments, compound 2.1 may be prepared as shown in FIG. 1. As shown in FIG. 1, carbonyl diimidazole-coupling of 2-(benzyloxy)benzoic acid, 2.2, with methyl anthranilic acid, 2.4, produces amide 2.3. The pyrolysis of amide 2.3 at 230° C., under vacuum produces benzoxazole 2.5. Benzoxazole 2.5 reacts with NaOH and THF at 60° C. to produce benzoxazole acid 2.6. Coupling benzoxazole acid 2.6 with methyl 2,3-diaminobenzoate, 2.7, yields amide 2.8. A final cyclodehydration and deprotection yields compound 2.1. Compound 2.1 is produced by heating amide 2.8 in the presence of p-toluenesulfonic acid (p-TsOH). Compound 2.1 may be produced with a 35% overall yield from 2-benzyloxybenzoic acid.

In an embodiment, the UK-1 analogue, compound 2.9, may be a simple benzimidazole analogue. In some embodiments, compound 2.9 may be prepared as shown in FIG. 2. As shown in FIG. 2, condensation of 2-benzyloxybenzoic acid, 2.2, with methyl 2,3-diaminobenzoate, 2.10, yielded amide 2.11 with a yield of about 76%. Amide 2.11 may be cyclized by heating in acetic acid at a temperature of about of about 120° C.-130° C. to give benzimidazole ester 2.12. Compound 2.9 may be produced by the hydrolysis of benzimidazole ester 2.12. Hydrolysis of the benzimidazole 2.9 may be performed by heating in the presence of NaOH and THF. Compound 2.9 may be produced with an overall yield of 49%.

The UK-1analogue may be a substituted benzoxazole, compound 2.13. In an embodiment, compound 2.13 may be produced by the method shown in FIG. 3. As shown in FIG. 3, Amide 2.3, produced by the method shown in FIG. 1, may be reacted with p-TsOH and toluene to yield compound 2.13. Compound 2.13 may be prepared by p-TsOH mediated cyclization and deprotection.

The UK-1analogue may be a benzlyated UK-1analogue, compound 2.15. Compound 2.15 may be synthesized by the method shown in FIG. 4. As shown in FIG. 4, when the cyclization/deprotection shown in FIG. 3 is performed under anhydrous conditions, compound 2.15 is produced as a side product. Compound 2.15 may be produced as a side product in the synthesis of UK-1. Compound 2.15 may arise from the interception of a benzyl cation formed by acid-mediated cleavage of the benzyl ester. In other methods of preparation of UK-1, the benzyl cation may be intercepted by adventitious water in the reaction mixture. Under more anhydrous conditions, the benzyl cation may be able to participate in Friedel-Crafts alkylation chemistry, producing compound 2.15. In an embodiment, UK-1analogue 2.15 may not be formed if the UK-1 synthesis of FIG. 4 takes place in the presence of 2-4 equivalents of water.

Cancer Cell Cytotoxicity and Antibacterial Activity of UK-1 Analogues

The cancer cell cytotoxicity and antibacterial activity of UK-1 analogues was examined in order to determine the structural basis for the unique cancer cell selective cytotoxicity of UK-1. Benzoxazole, 2.16, and 2-(2′-hydrophenyl)benzoazole, 2.17, were also examined.

Antibacterial and Anticancer Activity of UK-1 and Analogues.

Antibacterial IC₅₀ ^(a) Meth-resist Cytotoxicity Compound S. aureus S. aureus IC₅₀ Range^(b) UK-1 NA^(c) NA^(c) 0.32-65 μM 2.15 nd^(d) nd^(d) 4.5-27 μM 2.1 NA NA 70->100 μM 2.5 NA NA >100 μM 2.6   43 μM   29 μM >100 μM 2.9  102 μM  102 μM >100 μM 2.13 NA NA 0.88-9.1 μM 2.16 NA NA >100 μM 2.17 NA NA >100 μM Ciprofloxacin 0.45 μM 0.45 μM nd Mitomycin C nd nd 0.08-0.27 μM ^(a)Sample concentration that affords 50% growth of the test organism. ^(b)Range of IC₅₀ values obtained by alamarBlue cytotoxicity assays against MCF-7, HL-60, HT-29, and PC-3 cells after 72 h at 37° C. ^(c)No activity observed at concentrations up to 50 μg/mL. ^(d)Not determined.

The above table shows the results of cytotoxicity testing against MCF-7, HT-29, HL60, and PC-3 cell lines along with the antibacterial assays against S. aureus and a methicillin-resistant strain of S. aureus. UK-1 shows a wide range of cytotoxic activity against the four cancer cell lines. This cytotoxic activity may be due to the relative resistance of the HT-29 cells to the action of UK-1. The benzylated UK-1analogue, compound 2.15, demonstrates a diminished cytotoxic effect against the cancer cell lines relative to UK-1; however, the spectrum of activity of compound 2.15 is quite distinct. Compound 2.15 is more active than UK-1 against HT-29 cells (IC₅₀ 5.2 μM for compound 2.15 versus 65 μM for UK-1). Compound 2.15 is about ten-fold less potent against MCF-7 (IC₅₀ 13 μM for compound 2.15 versus IC₅₀ 1.6 μM for UK-1, respectively) and PC-3 cells (IC₅₀ 4.5 μM for compound 2.15 versus IC₅₀ 0.4 μM for UK-1, respectively). Compound 2.15 is twenty-fold less active than UK-1 against HL60 cells (IC₅₀ 27 μM for compound 2.15 versus 0.32 μM for UK-1). Compound 2.15 has different bacterial cell selectivity properties than UK-1.

The aza-analogue of UK-1, compound 2.1, does not exhibit anticancer activity against three of the four cell lines examined, with IC₅₀ values greater than approximately 100 μM against MCF-7, HT-29, and PC-3 cells. Compound 2.1 did demonstrate very modest activity against HL60 cells (IC₅₀=70 μM). Compound 2.1 may lack antibacterial activity against the two strains of S. aureus examined. Many other analogues of UK-1 also appeared to lack appreciable anticancer activity. Two analogues, compound 2.6 and compound 2.9, displayed some antibacterial activity against S. aureus strains. The activity of compounds 2.6 and 2.9 against S. aureus, while modest, is unexpected in light of the reported antibacterial activity of DMUK-1 and the apparent lack of antibacterial activity of methyl ester UK-1, compound 2.5, and the methyl ester of compound 2.4. Compound 2.17, 2-(2′-hydroxyphenyl)benzoxazole, is neither cytotoxic to cancer cells nor antibacterial, in conformity with previous reports of the lack of antibacterial activity for compound 2.17.

Compound 2.13 demonstrated cancer cell cytotoxicity. Compound 2.13 also displayed the selective activity against cancer cells versus bacterial cells that is characteristic of the natural product, UK-1. The anticancer activity of compound 2.13 appears to be less than UK-1against HL60 (IC₅₀=5.7 1M for compound 2.13 versus IC₅₀=0.4 μM for UK-1) and PC-3 cells (IC₅₀=0.88 μM for compound 2.13 versus IC₅₀=0.32 μM for UK-1). The anticancer activity for compound 2.13 appears to be equal to or better than UK-1against MCF-7 (IC₅₀=1.5 μM for compound 2.13 versus IC₅₀=1.6 μM for UK-1) and HT-29 cells (IC₅₀=9.1 μM for compound 2.13 versus IC₅₀=65 μM for UK-1). Compound 2.13 does not appear to be active against S. aureus, P. aeruginosa, Cryptococcus neoformans, or Candida albicans (IC₅₀ greater than 50 μg/mL)

Metal Binding of UK-1 Analogues

Association Constants for Metal Ion Binding by UK-1 and Analogues.

Compound Metal Ion UK-1 2.1 2.13 Mg²⁺ 2.0 × 10⁶ M⁻¹ 4.0 × 10⁴ M⁻¹ 3.2 × 10⁵ M⁻¹ Ca²⁺ 4.0 × 10⁴ M⁻¹ 1.0 × 10⁵ M⁻¹ 6.3 × 10³ M⁻¹ Zn²⁺ 2.0 × 10⁶ M⁻¹ 3.2 × 10⁶ M⁻¹ 1.3 × 10⁶ M⁻¹ Fe³⁺ 2.5 × 10⁵ M⁻¹ 1.0 × 10⁴ M⁻¹ 7.9 × 10³ M⁻¹

Previous work indicated the metal ion binding mechanism of UK-1, so the method of continuous variation was used to determine the ability of selected UK-1analogues to bind a variety of metal ions. Methanolic solutions of UK-1 or UK-1 analogues and metal ion salts [Ca(NO₃)₂, Mg(NO₃)₂, Zn(NO₃)₂, or Fe(NO₃)₃] at various molar ratios, and 20 μM combined total concentration, were prepared and the absorbance at 418 nm was determined. Plots of A_(418 nm) versus mole ratio of ligand demonstrated maxima at 0.5 mole ratio for all metal ions, indicating a 1:1 stoichiometry for each of the metal ion complexes. Compound 2.1, which appears to lack cancer cell cytotoxicity, does not bind Mg²⁺ and Fe³⁺ ions as well as UK-1. Compound 2.1 does bind with Ca²⁺ and Zn²⁺ better than UK-1.

Compound 2.13, which appears to have cancer cell cytotoxic properties, retains similar Zn²+ metal ion binding ability of UK-1. Compound 2.13 appears to have slightly diminished, relative to UK-1, Mg²⁺, Ca²⁺, and Zn²⁺, binding ability

An appropriate 4-substituent on the 2-(2′-hydroxyphenyl)benzoxazole core may be important for efficient Mg²⁺ binding ability. The 4-unsubstituted 2-(2′-hydroxyphenyl)benzoxazole, compound 2.17, does not appear to complex Mg²⁺ ions. Compound 2.17 does appear to form complexes with Zn²⁺.

The introduction of a 4-carbomethoxy substituent, as in compound 2.13, on the (2′-hydroxyphenyl)benzoxazole core imparts Mg²⁺ ion binding ability. A 4-(benzoxazo-2-yl) substituent, as in UK-1 and compound 2.1, on the (2′-hydroxyphenyl)benzoxazole core does not appear to impart Mg²⁺ ion binding ability. Mg²⁺ ion binding may play a role in cancer cell cytotoxicity of UK-1 analogues. Cytotoxic UK-1 and compound 2.13 may bind Mg²⁺ ions better than the less cytotoxic analogues, compounds 2.1 and 2.17. The selective activity of compound 2.13 against cancer cells, but not bacteria, may reflect a selective activity similar to UK-1. In some embodiments, compound 2.13 may be a minimum pharmacophore for the selective anticancer activity of UK-1.

The structural basis for Mg²⁺ ion binding and selective cytotoxicity of UK-1 may be the same. Both Mg²⁺ ion binding and selective cytotoxicity may require an appropriately 4-substituted 2-(2′-hydrophenyl)benzoxazole moiety. While it is unlikely that Mg²⁺ ion binding per se is the origin of the selective cytotoxicity of UK-1 and analogue 2.13, the ability of UK-1 to form metal ion complexes that can bind to DNA and inhibit DNA-processing enzymes indicates that Mg²⁺ ion binding by UK-1 may lead to biologically relevant complexes with a specific target in cancer cells.

As discussed above, certain non-covalent associations between UK-1 or UK-1 analogs and DNA are correlated with the selective cytotoxicity of these compounds towards cancer cell lines. In particular, metal-ion mediated complexation between UK-1 and UK-1 analogs with DNA is important in predicting the biological activity of the natural product. UK-1 and related analogs that are selectively cytotoxic, associate with double-stranded DNA more strongly in the presence of certain metal ions, such as Mg²⁺ Ni²⁺, Cu²⁺, Zn²⁺, or Co²⁺. In contrast, analogs of UK-1 that are antibacterial associate with double-stranded DNA in a metal ion-independent fashion. This later behavior is similar to that displayed by antibacterial gyrase-inhibiting drugs such as ciprofloxacin. Finally, UK-1 analogs that are neither antibacterial nor cytotoxic fail to bind to double-stranded DNA, regardless of the presence or absence of divalent metal ions. This discovery provides a rapid and convenient method to screen for compounds with potential anticancer activity (e.g., those that bind to DNA in a metal ion-dependent fashion) or compounds that have potential antibacterial use (e.g., those that bind to DNA in a metal ion-independent fashion), and to distinguish these compounds from those that are not antibacterial or anticancer (e.g., those that do not bind to DNA in the presence or absence of metal ions).

Cancer Cell Cytotoxicity, Antibacterial Activity, and DNA Binding Ability of UK-1 and Analogs Staph. Cyto- DNA- aureus ^(a) toxicity^(b) Binding IC₅₀ IC₅₀ ESI-MS^(c)

>50μg/mL 0.32-65μM MetalDepend.^(d)

>50μg/mL >100 μM None^(e)

>50μg/mL 0.88{hacek over (G)}9.1 μM MetalDepend.^(d)

29 μg/mL >100 μM MetalIndepend.^(f) Ciprofloxacin 0.45μg/mL >100 μM MetalIndepend.^(f) ^(a)concentration that inhibits the growth of Methacilline-resistant Staph. Aureus by 50%. ^(b)range of concentrations required to inhibit the growth of MCF-7, HT-29, and PC-3 cells by 50% as determined by the alamarBlue assay. ^(c)formation of ligand-DNA complex ions in the ESI-MS of ligand and double-stranded DNA oligonucleotide solutions in the presence or absence of added NiCl₂. ^(d)all ligand-DNA complex ions also contained a metal ion. ^(e)no ligand-DNA complex ions observed. ^(f)ligand-DNA and ligand-DNA-metal ion complex ions observed.

We have also found that UK-1can cleave DNA in the presence of oxidizing agents or in the presence of oxidizing agents and metal ions. Supercoiled ΦX174 DNA (50 μM) was incubated for 24 hrs in pH 8 Tris buffer (10 mM) at 37° C. with UK-1 (15-25 μM), H₂O₂ (5 μM-5 mM), or NiCl₂ (15-25 μM), or a combination of these agents.

Agarose gel electrophoresis was carried out to resolve the DNA products. There was no evidence of DNA cleavage in the samples containing only NiCl₂ or UK-1. There was an increase in the proportion of nicked, relaxed DNA product in the presence of an oxidant (e.g., H₂O₂), but no linear DNA that would indicate double-stranded DNA cleavage. However, in the presence of both UK-1 and an oxidant (e.g., H₂O₂), there was significantly more DNA cleavage, and linear DNA products were observed, indicating both single- and double-stranded DNA cleavage. The combination of UK-1, H₂O₂, and NiCl₂ was particularly active in inducing DNA cleavage, affording more relaxed and linear DNA products than control reactions that contained only H₂O₂ and NiCl₂.

FIG. 5 depicts cleavage of supercoiled DNA by UK-1 in the presence of oxidizing agents and metal ions. Supercoiled ΦX174 DNA (50 μM bp) was incubated with UK-1 (23 μM), NiCl₂ (23 μM), H₂O₂ (5 mM), or combinations of these agents at 37° C. for 24 h and the DNA products subjected to agarose gel electrophoresis with ethidium bromide staining. Lanes: C, untreated supercoiled DNA; 1, DNA+UK-1; 2, DNA+NiCl₂; 3, DNA+H₂O₂; 4, DNA+UK-1+NiCl₂; 5, DNA+UK-1+H₂O₂; 6, DNA+UK-1+NiCl₂+H₂O₂; 7, DNA+NiCl₂+H₂O₂.

EXAMPLES

General. All reagents and solvents were purchased from Aldrich Chemical Company and used without further purification, unless noted. THF was distilled from sodium/benzophenone immediately prior to use. CH₂Cl₂ and DMF were distilled from CaH₂ immediately prior to use. Toluene was distilled from sodium metal. UV/V is spectra for the continuous variation plots were determined on a Varian Cary 3E spectrophotometer. Melting points (open capillary) are uncorrected. Unless otherwise noted, ¹H and ¹³C NMR spectra were determined in CDCl₃ on a spectrometer operating at 300 and 75.5 MHz, respectively. All mass spectra were obtained by chemical ionization using methane as the ionizing gas. Chromatography refers to flash chromatography on silica gel, and Rf values were determined using silica gel-GF TLC plates (Merck) using the solvent system indicated.

Compound 2.3: Methyl 2-{[2-(benzyloxy)benzoyl]amino}-3-hydroxybenzoate. Carbonyldiimidazole (CDI) (5.10 g, 31.5 mmol) was dissolved in 50 ml dry THF with stirring at room temperature under an argon atmosphere, and 2-(benzyloxy)benzoic acid (compound 2.4) (7.18 g, 31.5 mmol) was added to the mixture. After the evolution of CO₂ ceased (ca. 5 min) the reaction mixture was stirred for an additional 10 minutes and methyl 2-amino-3-hydroxybenzoate (3.5 g, 21 mmol) was added. After stirring for an additional 10 minutes at room temperature, the reaction mixture was heated under reflux for 18 hours. The reaction mixture, brown in color, was concentrated and dissolved in a minimum volume of ethyl acetate (EtOAc). Silica gel (60-100 mesh) was added to make a slurry and solvent was evaporated to dryness. Column chromatography was performed using 20% EtOAc in hexane to give a light yellow solid (6.9 g, 87%): mp 104-105° C.; R_(f) 0.328 (20% EtOAc in hexanes); ¹H NMR δ 3.80 (s, 3H), 5.50 (s, 2H), 7.05 (d, 1H, J=8.1 Hz), 7.10 (t, 1H), 7.24 (m, 2H), 7.34 (m, 3H), 7.47 (m, 3H), 7.61 (d, 1H, J=7.8 Hz),8.27 (d, 1H, J=9.0 Hz), 9.28 (brs, 1H, NH), 12.27 (s, 1H, OH); ¹³C NMR δ 52.20, 70.80, 113.34, 120.94, 121.27, 121.58, 122.97, 125.70, 126.05, 126.93, 128.01, 128.21, 128.60, 132.69, 133.93, 136.22, 150.76, 157.01, 165.51, 167.57; CIMS m/z 378 (MH⁺); HRMS m/z calc'd for C₂₂H₂₀NO₅: 378.1341, found 378.1343.

Compound 2.5: Methyl 2-[2-(benzyloxy)phenyl]-1,3-benzoxazole-4-carboxylate. Compound 2.3 (6.9 g, 1.83 mol) was heated neat at 230° C. for 2 hours with stirring in a long neck 50 ml round bottomed flask under vacuum, which was applied slowly at small intervals (20 minutes) to remove the water vapor generated in the reaction. Upon cooling, a light orange solid mass was obtained, which was dissolved in a minimum volume of EtOAc. To this solution, hexane was added with stirring to precipitate the product benzoxazole, which was filtered to give a white solid (6.0 g, 91%): mp 100-102° C.; R_(f) 0.468 (40% EtOAc in hexanes); ¹H NMR δ 3.99 (s, 3H), 5.28 (s, 2H), 7.07 -7.13 (m, 2H), 7.27-7.33 (m, 1H), 7.35-7.42 (m, 3H), 7.48 (ddd, 1H, J=7.8, 1.7 (br d, 1H), 7.73 (dd, 1H, J=8.1, 1.0 Hz), 8.02 (dd, 1H, J=7.8,1.2,1.0 Hz), 8.25 (br d, 1H); ¹³C NMR δ 52.40, 70.56, 113.61, 114.67, 116.31, 121.03, 121.98, 124.12, 126.79 (2C), 127.65, 128.45, 131.92, 133.19, 136.66, 141.46, 151.24, 157.74, 163.60, 165.97; CIMS m/z 360 (MH⁺); HRMS m/z calc'd for C₂₂H₁₈NO₄: 360.1235, found 360.1228.

Compound 2.6: 2-[2-(benzyloxy)phenyl]-1,3-benzoxazole-4-carboxylic acid. Compound 2.5 (6.0 g, 1.67 mol) was dissolved in 80 ml of THF and 40 ml of 5M NaOH was added. The reaction mixture was stirred at 60° C. with stirring for 2 hours. The reaction mixture was cooled to room temperature, diluted with EtOAc and acidified with concentrated HCl. The organic layer was washed with brine two times and dried over anhydrous Na₂SO₄. The solvent was evaporated and the residue was dried under vacuum to afford a white solid that was recrystallized from EtOAc and hexanes (5.2 g, 90%): mp 103-104° C.; R_(f) 0.15 (40% EtOAc in hexanes); ¹H NMR δ 5.28 (s, 3H), 7.12-7.20(m, 2H), 7.37-7.49 (m, 4H), 7.52-7.60 (m, 3H), 7.77 (dd, 1H, J=7.9, 0.9 Hz), 8.14 (dd, 1H, J=7.6, 1.2 Hz), 8.21 (dd, 1H, J=7.8, 1.5 Hz); ¹³C NMR δ 70.98, 113.76, 114.61, 115.28, 120.07, 121.27, 125.48, 127.08, 127.31, 128.39, 128.90, 131.72, 134.44, 136.09, 141.13, 149.90, 158.27, 163.16, 165.00; CIMS m/z 346 (MH⁺); HRMS m/z calc'd for C₂₁H₁₆NO₄: 346.1079, found 346.1078.

Compound 2.8: Methyl 2-amino-3-({[2-(2-benzyloxyphenyl)-1,3-benzoxazol-4-yl]carbonyl}aminobenzoate. To a solution of compound 2.6 (1.035 g, 3 mmol) in 40 ml of dry CH₂Cl₂ was added 10 ml of freshly distilled oxalyl chloride. After stirring for 10 minutes, 5 drops of DMF was added to the reaction mixture. The reaction mixture was stirred for 2 hours at room temperature and the solvent was removed by evaporation. The resultant yellow solid was dried under vacuum for 3 hours. The yellow solid was then dissolved in 15 ml of dry CH₂Cl₂ and transferred via canula to a solution of methyl 2,3-diaminobenzoate (compound 2.7)(498 mg, 3 mmol) dissolved in 20 ml of dry CH₂Cl₂ and 2 ml of pyridine. The reaction mixture was stirred at room temperature overnight and then diluted with CH₂Cl₂. The solution was washed with 1% HCl, satd. aq. NaHCO₃ and satd. aq. NaCl. The organic layer was dried over anhydrous Na₂SO₄ and the solvent was evaporated to give the residue which was recrystallized from EtOAc to afford a light yellow solid (1.077 g, 73%): mp 178° C.; R_(F) 0.447 (40% EtOAc in hexanes); ¹H NMR δ 3.91 (s, 3H, CH₃), 5.30 (s, 2H, CH₂), 6.23 (brs, 2H, NH₂), 6.75 (t, 1H, J=7.9 Hz),7.14-7.30 (m, 6H), 7.45-7.58 (m, 4H), 7.77 (dd, 1H, J=8.1, 0.7 Hz), 7.80 (dd, 1H, J=8.1, 1.5 Hz), 7.93 (dd, 1H, J=7.6, 1.2 Hz), 8.24 (dd, 1H, J=8.0, 1.7, 1.5 Hz), 8.28 (dd, 1H, J=7.6, 0.7 Hz), 10.64(s, 1H, NH); ¹³CNMR 651.59,70.89, 111.92, 113.94, 113.97, 115.46, 115.77, 121.28, 123.82, 124.74, 125.02, 125.87, 1126.83, 127.81, 128.23, 128.41, 129.37, 131.55, 133.75, 136.12, 139.45, 139.45, 144.36, 150.33, 157.87, 162.77, 162.82, 168.64; CIMS m/z 494 (MH⁺); HRMS m/z calc'd for C₂₉H₂₄N₃O₅: 494.1715, found 494.1711.

Compound 2.1: Methyl 2-[2-(2-hydroxyphenyl)-1,3-benzoxazol-4-yl]-1H-benz imidazole-4-carboxylate. Compound 2.8 (493 mg, 1 mmol) was dissolved in 20 ml of toluene and p-toluenesulfonic acid monohydrate (475 mg, 2.5 mmol) was added. The reaction mixture was refluxed for 2 hours and the solvent was distilled off. The residue was treated with CH₂Cl₂ and the solution was washed with satd. aq. NaHCO₃ and satd. aq. NaCl. The organic layer was dried over anhydrous Na₂SO₄ and the solvent was evaporated to give a solid, The solid was with EtOAc and filtered. The residue was dried and purified by preparative TLC using 40% EtOAc in hexanes to afford a white solid (200 mg, 68%): mp 243° C.; R_(F) 0.170 (40% EtOAc in hexanes); ¹H NMR δ 4.20 (s, 3H, CH₃), 7.10 (t, 1H, J=8.1, 7.2 Hz), 7.24 (d, 1H, J=8.4 Hz), 7.41 (t, 1H, J=7.8, 7.5 Hz), 7.52-7.61 (m, 2H), 7.74 (d, 1H, J=7.5 Hz), 8.02 (d, 1H, J=7.8 Hz), 8.09-8.14 (m, 2H), 8.56 (d, 1H, J=7.2 Hz), 10.53 (s, 1H, NH), 11.95 (brs, 1H, OH); ¹³C NMR δ 52.65, 109.97, 112.49, 113.88, 117.84, 119.20, 120.19, 122.61, 124.30, 125.71, 126.08, 127.87, 134.10, 134.45, 134.54, 137.65, 142.83, 149.23, 149.39, 158.54, 163.96, 166.64; CIMS m/z 386(MH⁺); HRMS m/z calc'd for C₂₂H₁₆N₃O₄: 386.1141, found 386.1150.

Compound 2.11: Methyl 2-amino-3-{[2-(benzyloxy)benzoyl]amino}benzoate. Carbonyldiimidazole (compound 2.10) (2.43 g, 15 mmol) was dissolved in 30 ml dry THF with stirring under argon at room temperature and 2-(benzyloxy)benzoic acid (compound 2.2) (3.42 g, 15 mmol) was added to the mixture. Heavy evolution of CO₂ was observed for 3-4 min. After stirring for 10 minutes, methyl 2,3-diaminobenzoate (1.66 g, 10 mmol) was added and stirring continued further for 10 minutes. The reaction mixture was then heated under reflux for 5 days. The reaction mixture, dark brown in color, was concentrated and dissolved in a minimum volume of EtOAc. Silica gel (60-100 mesh) was added to make a slurry and the solvent was evaporated. The resulting solid was added to the top of a dry-packed chromatography column that was eluted with 20% EtOAc in hexane to give the product as a white solid (2.86 g, 76%): mp 111-113° C.; R_(F) 0.373 (40% EtOAc in hexanes); ¹H NMR δ 3.86 (s, 3H, CH₃), 5.25 (s, 2H, CH₂),5.58 (s, 2H, NH₂), 6.66 (t, 1H, J=8.0 Hz), 7.13-7.18 (m, 2H), 7.43-7.58 (m, 7H),7.75 (d, 1H, J=7.8 Hz), 8.27 (d, 1H, J=7.8 Hz), 9.20 (brs, 1H, NH); ¹³C NMR δ 51.68, 71.72, 112.57, 112.59, 116.10, 121.75, 121.83, 124.76, 128.49, 128.77, 129.12 (2C), 130.09, 132.72, 133.32, 135.01, 144.51, 156.54, 163.98, 168.40; CIMS m/z 377 (MH⁺); HRMS m/z calc'd for C₂₂H₂₁N₂O₄: 377.1501, found 377.1499.

Compound 2.12: Methyl 2-[2-(benzyloxy)phenyl]-1H-benzimidazole-4-carboxylate. Compound 2.11 (2.86 g, 76 mmol) was heated in glacial acetic acid (20 ml) at 120° C. for 30 minutes with stirring in a 50 ml round bottom flask. The reaction mixture was allowed to cool and then poured into ice cold water and extracted with CH₂Cl₂. The combined organic layers were washed with satd. aq. NaHCO₃, brine and dried (Na₂SO₄). The solvent was removed by evaporation and the residue was purified by chromatography on silica gel (60-100 mesh) using 20 to 40% EtOAc in hexane to afford benzimidazole ester 8 as a white solid (2.3 g, 80%): mp 100-101° C.; R_(F) 0.412 (40% EtOAc in hexanes); ¹H NMR δ 3.67 (s, 3H, CH₃), 5.36 (s, 2H, CH₂), 7.17-7.23 (m, 2H), 7.33 (t, 1H, J=7.7 Hz), 7.40-7.49 (m, 4H), 7.56 (dd, 2H, J=7.6, 1.7 Hz), 7.90 (dd, 1H, J=7.6, 1.0 Hz),8.03 (d, 1H, J=8.4 Hz), 8.62 (dd, 1H, J=7.8, 1.8 Hz), 11.56 (brs, 1H); ¹³C NMR δ 51.69, 71.29, 112.95, 113.40, 117.93, 121.68, 121.90, 124.30, 124.52, 127.95, 128.51, 128.92, 130.51, 131.58, 134.16, 135.64, 143.74, 150.91, 156.33, 166.33; CIMS m/z 359 (MH⁺); HRMS m/Z calc'd for C₂₂H₁₉N₂O₃: 359.1395, found 358.1393.

Compound 2.9: 2-[2-(benzyloxy)phenyl]-1H-benzimidazole-4-carboxylic acid. Compound 2.12 (1.0 g, 2.78 mmol) was dissolved in 20 ml of THF and 8 ml of 5M NaOH was added. The reaction mixture was stirred at 60° C. for 3 hours. The reaction mixture was allowed to cool to room temperature and acidified with concentrated HCl. The obtained white solid was filtered, washed with water, and dried under vacuum to afford the acid 9 (780 mg, 81%): mp 188° C.; R_(f) 0.125 (40% EtOAc in hexanes); ¹H NMR (DMSO-d₆) δ 5.32 (s, 2H, CH₂), 7.15 (t, 1H, J=7.5), 7.29-7.41 (m, 5H), 7.49 (t, 1H, J=8.1 Hz), 7.56 (d, 2H, J=7.4 Hz), 7.78 (d, 1H, J=7.7 Hz), 7.92 (d, 1H, J=7.5 Hz), 8.32 (dd, 1H, J=7.9, 1.8 Hz), 11.85 (brs, 1H); ¹³C NMR (DMSO-d₆) δ 70.27, 107.28, 114.01, 117.73, 121.49, 121.66, 124.21, 123,68, 127.85, 128.12,128.64,130.00, 131.80, 134.68, 136.13, 141.02, 150.23, 155.89, 167.11; CIMS m/z 345 (MH⁺); HRMS m/z calc'd for C₂₂H₁₇N₂O₃: 345.1239, found 345.1233

Compound 2.13: Methyl 2-(2-hydroxyphenyl)-benzoxazole-4-carboxylate. Compound 2.3 (103 mg, 0.3 mmol) was dissolved in 4 ml of toluene and p-toluenesulfonic acid monohydrate (142 mg, 0.75 mmol) was added. The reaction mixture was refluxed for 1.5 hours and, after cooling to room temperature, was diluted with EtOAc. The organic layer was washed with satd. aq. NaHCO₃ and satd. aq. NaCl, dried over anhydrous Na₂SO₄, and the solvent was evaporated to afford the benzoxazole as a solid (45 mg, 58%): mp 134-135° C.; R_(F) 0.578 (30% EtOAc in hexanes); ¹H NMR δ 4.05 (s, 3H, CH₃), 7.01 (t, 1H), 7.13 (d, 1H, J=8.1 Hz), 7.4-7.5(m, 2H), 7.78 (d, 1H, J=8.1 Hz), 8.01 (d, 1H, J=7.5 Hz), 8.06 (d, 1H, J=8.1 Hz); ¹³C NMR δ 52.38, 109.91, 114.86, 117.63, 119.55, 121.24, 124.69, 127.15, 127.40, 134.19, 139.36, 149.70, 159.33, 164.26, 165.52; CIMS m/z 270 (MH⁺); HRMS m/z calc'd for C₁₅H₁₂NO₄: 270.0766, found 270.0766.

Compound 2.14: Methyl 3-hydroxy-2-{[2-(benzyloxyphenyl)-1,3-benzoxazol-4-yl]carbonyl}aminobenzoate. To a solution of compound 2.6 (3.45 g, 10 mmol) in 60 ml of dry CH₂Cl₂ was added 10 ml of freshly distilled oxalyl chloride. The reaction mixture was stirred for 10 minutes and 0.3 ml of dimethyl formamide was added to initiate the reaction. The reaction mixture was stirred for 2 hours at room temperature and the solvent was removed by evaporation. The resultant yellow solid was dried under vacuum for 3 hours. The yellow solid was then dissolved in 20 ml of dry CH₂Cl₂ and transferred via cannula to a solution of methyl 2-amino-3-hydroxybenzoate (1.67 g, 10 mmol) dissolved in 40 ml of dry CH₂Cl₂ and 3 ml of pyridine. The reaction mixture was stirred at room temperature overnight and then diluted with dichloromethane. The solution was washed with 1% HCl, satd. aq. NaHCO₃ and satd. aq. NaCl. The organic layer was dried over anhydrous Na₂SO₄ and the solvent was evaporated to give a residue which was subjected to column chromatography using CH₂Cl₂ as eluent to afford a light yellow solid (3.8 g, 77%): mp 157-158° C.; R_(f) 0.329 (CH₂Cl₂); ¹H NMR δ 3.79 (s, 3H), 5.29 (s, 2H), 7.13-7.39 (m, 8H), 7.50-7.55 (m, 4H), 7.65 (dd, 1H, J=7.5, 1.5 Hz), 7.80 (brd, 1H, J=8.1 Hz), 8.31 (brd, 1H, J=7.8 Hz), 8.50 (dd, 1H, J=7.5, 1.8 Hz); ¹³C NMR δ 52.23, 70.71, 113.76, 114.71, 115.76, 121.18, 122.51, 122.98, 123.62, 124.73, 125.62, 126.35, 126.50, 126.69, 127.68, 127.87, 132.31, 133.66, 136.28, 139.91, 150.87, 151.20, 157.89, 163.46, 164.40, 167.11; CIMS m/z 495 (MH⁺); HRMS m/z calc'd for C₂₉H₂₃N₂O₆: 495.1556, found 495.1553.

Compound 2.15: Methyl 2′-(3-benzyl-2-hydroxyphenyl)-2,4′-bi-1,3-benzoxazole-4-carboxylate. A mixture of dry toluene (20 ml) and p-toluenesulfonic acid monohydrate (950 mg, 5 mmol) was refluxed under a reflux condenser. Compound 2.14 (988 mg, 2 mmol) was added and the reaction mixture was refluxed for an additional 2 hours. After the solvent was removed by distillation, the residue was dissolved in CH₂Cl₂ and the solution was washed with satd. aq. NaHCO₃ and satd. aq. NaCl. The organic layer was dried over anhydrous Na₂SO₄ and the solvent was evaporated to give 432 mg of solid that was a mixture of UK-1 and compound 2.15 (TLC and ¹H NMR) in the ratio of 1.6:1.0. Column chromatography using 5 to 20% EtOAc in hexane afforded 10 mg pure compound 2.15 (1%) along with 300 mg UK-1 (13%) and a large mixed fraction (122 mg). Compound 2.15: mp 204-205° C.; R_(f) 0.357 (30% EtOAc in hexanes); ¹H NMR δ 4.09 (s, 3H, CH₃), 4.15 (s, 2H, CH₂), 6.96 (t, 1H, J=7.6 Hz, C₅—H),7.20-7.14 (m, 1H, C₃-benzyl-H(para)), 7.26 (s, 2H, C₃-benzyl-H(meta)), 7.27 (s, 2H, C₃-benzyl-H(ortho)), 7.30 (dd, 1H, J=7.5, 1.2 Hz, C₄—H), 7.46 (t, 1H, J=7.9 Hz, C₆—H), 7.52 (t, 1H, J=8.0 Hz, C₆′-H), 7.77 (dd, 1H, J=8.1, 1.0 Hz, C₇′-H), 7.86 (dd, 1H, J=8.1, 1.0 Hz, C₇—H), 7.96 (dd, 1H, J=7.9, 1.6 Hz, C₆—H), 8.10 (dd, 1H, J=7.7, 1.0 Hz, C₅—H), 8.31 (dd, 1H, J=7.8, 0.9 Hz, C₅′-H); ¹³C NMR δ 35.83, 52.63, 109.72, 113.82, 115.09, 117.61, 119.33, 122.57, 124.93, 125.11, 125.24, 125.51, 126.07, 127.57, 128.42, 128.81, 129.77, 135.26, 138.76, 140.38, 141.56, 150.01, 151.22, 157.65, 161.71, 165.05, 166.32: CIMS m/z 477 (MH⁺); HRMS m/z calc'd for C₂₉H₂₁N₂O₅: 477.1450, found 477.1442.

Compound 2.16: Methyl 2-(2-Hydroxy-phenyl)-benzooxazole-7-carboxylate. To a solution of 2-benzyloxybenzoic acid dry CH₂Cl₂ was added to 1 ml of freshly distilled oxalyl chloride. After stirring for 10 minutes, 1 drop of DMF was added to the reaction mixture. The reaction mixture was stirred for 2 hours at room temperature and the solvent was removed by evaporation. The resultant yellow oil was dried under vacuum for 3 hours. The yellow oil was then dissolved in 5 ml of dry xylenes and transferred via cannula to a solution of 3-aminosalacylic acid (g, 31.5 mmol) in xylenes. The mixture was stirred at room temperature for 1 h, then heated to reflux overnight. After the reaction mixture had cooled to room temperature, p-toluenesulfonic acid monohydrate (475 mg, 2.5 mmol) was added and the mixture heated to reflux for an additional 12 h. The reaction mixture was cooled to 0° C. and treated with an excess of TMSCHN₂. The product was purified by column chromatography (20% EtOAc in hexane) to give a light yellow solid (42 mg, 36%): ¹H NMR δ 4.04 (s, 3H, CH₃), 7.00 (m, 1H), 7.10 (d, 1H, J=8.4 Hz), 7.4-7.5 (m, 2H), 7.87 (dd, 1H, J=8.1, 1.2 Hz), 7.97 (dd, 1H, J=7.5, 1.2 Hz), 8.06 (dd, 1H, J=8.1, 1.2 Hz); CIMS m/z 270 (MH⁺); HRMS m/z calc'd for C₁₅H₁₂NO₄: 270.0763, found 270.0766.

Cytotoxicity Assays. Cell culture cytotoxicity assays were performed using the. alamarBlue method, as described by Ahmed et al., J. Immun. Methods 1994, 170, 211-224, which is incorporated herein by reference. Briefly, aliquots of 100 μL of cell suspension (1.0-2.5×10³ cells) was placed in 96-well microtiter plates in an atmosphere of 5% CO₂ at 37° C. After 24 hours, 100 μL of culture medium and 2 μL of compound in vehicle (culture media with 40% pyridine) or vehicle alone were added, and the plates incubated an additional 72 hours. The final pyridine concentration in all cases was 0.4%; at this pyridine concentration there was no affect on the growth of cells compared to cells in culture media without added pyridine. Compounds, along with mitomycin C as positive control, were evaluated in duplicate at final concentrations ranging from 0.001 to 100 μM. After the culture media had been removed from each well, 200 μL of fresh media and 20 μL of 90% alamarBlue reagent were added, followed by an additional 6 h incubation. The fluorescent intensity was measured using a Spectrafluor Plus plate reader with excitation at 530 nm and emission at 590 nm. Results are reported as IC₅₀ values, the average concentration required to produce a decrease of fluorescent intensity of 50% relative to vehicle-treated controls in two separate determinations.

Antimicrobial Bioassay: Staphylococcus aureus ATCC 29213 and methicillin-resistant S. aureus ATCC 43300 (MRS) were obtained from the American Type Culture Collection (Rockville, Md.) and stored on agar slants at 4° C. until needed. Susceptibility testing was performed using a modified version of the NCCLS methods: “Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria that Grow Aerobically,” M7-A4, Vol. 17, No. 2, NCCLS, (1997) and “Reference Method for Broth Dilution Antifungal Susceptibility Testing of Yeasts; Approved Standard,” M27-A, Vol. 17, No. 9, NCCLS, (1997), both of which are incorporated herein by reference. Microorganisms were subcultured prior to the assay by suspending cells from the slant in Eugon broth (BBL, Maryland) and incubating for 24 hours at 37° C. Inocula were prepared by diluting the subcultured organism in its incubation broth after comparison to the 0.5 McFarland Standard (a BaSO₄ suspension) to afford final inocula ranges of S. aureus: 1.0-5.0×10⁵ and MRS: 0.2-6.0×10⁵ CFU/ml. Test compounds were dissolved in DMSO, serially-diluted using normal saline, and transferred in duplicate to 96 well flat-bottomed microtiter plates. The microbial inocula were added to the samples to achieve a final volume of 200 μl and final compound concentrations starting with 50 μg/ml. Drug [Ciprofloxacin (ICN Biomedicals, Ohio)] as well as growth and blank (media only) controls were added to each test plate. Plates were read at 630 nm using the EL-340 Biokinetics Reader (Bio-Tek Instruments, Vermont) prior to and after incubation at 37° C. for 24 hours. Percent growth was calculated and plotted versus concentration to afford the IC₅₀, or sample concentration that affords 50% growth of the organism relative to controls.

Continuous variation plots. A 100 μM solution of UK-1 or analogue and 100 AM solutions of Ca(NO₃)₂, Mg(NO₃)₂, Zn(NO₃)₂, or Fe(NO₃)₃ were prepared in methanol. From these stock solutions, 15 samples (1 mL) of varying mole fraction of the metal ion were prepared at a constant, combined concentration of 20 μM for the ligand and the metal ion. The change in absorbance was monitored from 500 to 200 nm for each sample using as a reference a 1 mL solution containing the same concentration of ligand as the sample being measured but without the metal ion. The maximum absorbance change was typically around 418 nm but varied somewhat for each metal ion. The absorbance at 418 nm was plotted as a function of the mole fraction of the metal ion and the maximum absorbance change was determined. The absorbance of a sample containing the metal ion concentration corresponding to this maximum change and a 15 fold excess of metal ion at this ligand concentration, subtracted from a reference containing the same concentration of ligand but no metal ion was used to normalize the curve and obtain the conditional formation constant as described in the paper to Likussar, W.; Boltz, D. F. Anal. Chem. 1971, 43, 1265-1272, which is incorporated herein by reference.

While the present invention has been described with reference to particular embodiments, it will be understood that the embodiments are illustrated and that the invention scope is not so lirnited. Any variations, modifications, additions and improvements to the embodiments described are possible. These variations, modifications, additions and improvements may fall within the scope of the invention as detailed within the following claims. 

1. A method of synthesizing a chemical compound comprising a cyclic ring having the structure II

where R¹ is alkyl or aryl; where each R² is independently hydrogen, alkyl, aryl, or halogen; where n is 1 to 4; and where X is N and Y is O, comprising: reacting an acyclic compound haying the structure III with an acid in a hydrocarbon solvent;

where R¹ and R² are independently hydrogen, alkyl, aryl, or halogen; where n is 1 to 4; and where X and Y are independently N or O; heating the reaction.
 2. The method of claim 1, wherein the acid is an organic acid.
 3. The method of claim 1, wherein the acid is p-TsOH.
 4. The method of claim 1, wherein the solvent is an organic solvent.
 5. The method of claim 1, wherein heating the reaction comprises a temperature above 80° C.
 6. The method of claim 1, further comprising cooling the reaction down to at least room temperature and filtering off the acid. 